虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用

doi:10.3969/j.issn.1004-1524.2021.06.04

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (6): 993-1000.DOI: 10.3969/j.issn.1004-1524.2021.06.04

虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用

沈卫锋(), 郭琦, 刘莉, 牛宝龙, 翁宏飚, 楼宝^*()

浙江省农业科学院水生生物研究所,浙江杭州 310021

收稿日期:2020-04-20 出版日期:2021-06-25 发布日期:2021-06-25
通讯作者: 楼宝
作者简介:*楼宝,E-mail: loubao6577@126.com
沈卫锋(1977—),男,浙江桐乡人,博士,副研究员,主要从事水产病害研究。E-mail: shenwf@zaas.ac.cn
基金资助:
浙江省农业科学院新建学科经费

Cloning, expression and application in detection of SWP2 gene in Enterocytozoon hepatopenaei

SHEN Weifeng(), GUO Qi, LIU Li, NIU Baolong, WENG Hongbiao, LOU Bao^*()

Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2020-04-20 Online:2021-06-25 Published:2021-06-25
Contact: LOU Bao

摘要/Abstract

摘要：

虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)是虾类养殖过程中一种非常重要的病原微生物。目前常用的EHP检测方法是以核糖体18S基因为靶基因的PCR检测,但由于18S基因的高度保守性,该方法存在一定的非特异性扩增问题。本研究利用电子克隆方法,得到了特异性较高的EHP孢壁蛋白2(spore wall protein 2,SWP2)基因。该基因的cDNA全长为1 019 bp,开放阅读框全长687 bp,编码229个氨基酸,预测相对分子质量为26.32 ku,5'非编码区为120 bp,3'非编码区为179 bp,系统进化分析表明与毕氏肠孢虫和绒螯蟹肠孢虫具有较近的亲缘关系。构建了重组表达载体pET15b-SWP2,利用原核表达得到SWP2蛋白,相对分子质量约为28 ku,并制备了该蛋白的单克隆抗体。采用常规PCR及Western blot方法对市售的商品虾进行了EHP感染情况检测,结果显示,两种方法均能准确地进行检测分析。本研究结果为虾农肝肠微孢子虫病的诊断提供了技术支撑,同时为SWP2蛋白功能的研究打下了基础。

关键词: 虾肝肠胞虫, SWP2基因, 蛋白表达, 单克隆抗体, 病害检测

Abstract:

The Enterocytozoon hepatopenaei(EHP) from shrimp is a very important pathogenic microorganism in aquaculture. In present, the common method for EHP detection is PCR reaction which use ribosomal 18S gene as a target. The problem with this method is nonspecific amplification because of high conservatism of 18S gene. In this study, the spore wall protein 2 (EHPSWP2) was cloned with silico cloning method. The length of the cDNA of the SWP2 was 1 019 bp containing an open reading frame of 687 bp encoding a protein with 229 amino acids with predicted molecular weight of 26.32 ku, and 5’ UTR is of 120 bp and 3’ UTR is of 179 bp. The amino acids sequences and phylogenetic tree analysis showed that there is a close relationship among EHP, Enterocytozoon bieneusi and Hepatospora eriocheir. The recombinant expression vector pET15b-SWP2 was constructed for SWP2 protein expression, the molecular weight was 28 ku and the monoclonal antibodies against SWP2 protein were produced. The methods of PCR and western blot method were applied for EHP detection with Macrobrachium rosenbergii samples. This study provided a technical guarantee for the diagnosis of EHP disease by farmers, and laid the foundation for SWP2 protein research.

Key words: Enterocytozoon hepatopenaei, SWP2, protein expression, monoclonal antibody, disease detection

中图分类号:

S945.4⁺1

沈卫锋, 郭琦, 刘莉, 牛宝龙, 翁宏飚, 楼宝. 虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用[J]. 浙江农业学报, 2021, 33(6): 993-1000.

SHEN Weifeng, GUO Qi, LIU Li, NIU Baolong, WENG Hongbiao, LOU Bao. Cloning, expression and application in detection of SWP2 gene in Enterocytozoon hepatopenaei[J]. Acta Agriculturae Zhejiangensis, 2021, 33(6): 993-1000.

图/表 7

表1 引物序列

Table 1 Sequence of primers

引物名称 Primers	序列 Sequence(5'-3')	目的片段长度 Length of target fragment/bp
SWP2-F	CAAGAACAACCCACGAAGCAAG	568
SWP2-R	CCCTCATCACCATTGCTTACAG
18S-F	CGAACTACCTGGTAGCTGGTTC	801
18S-R	GACTTTCCAGAGCCTCCATACC

图1 EHP-SWP2基因cDNA全长序列及其编码蛋白氨基酸序列

Fig.1 Complete nucleotide sequences and deduced amino acid sequences of the EHP-SWP2 gene

图2 邻接法构建的基于虾肝肠微孢子虫EHP-SWP2蛋白与其他微孢子虫孢壁蛋白氨基酸序列的系统进化树

Fig.2 Phylogenetic tree of EHP-SWP2 protein and SWP protein from other microsporidia based on amino acid sequences with neighbor-joining method

图3 EHP-SWP2基因克隆与重组表达 A,SWP2基因片段PCR产物;B,重组质粒酶切;C,重组蛋白的Western验证;D,SDS-PAGE 鉴定纯化的His-SWP2重组蛋白。

Fig.3 Cloning and expression of EHP-SWP2 gene A, PCR fragment of SWP2 gens; B, Recombinant plasmid digestion; C, Western verification of recombinant protein; D, Verification of His-SWP2 protein with SDS-PAGE.

表2 不同细胞株分泌的单克隆抗体的效价和浓度检测结果

Table 2 Titer and concentration of monoclonal antibodies secreted by different cell lines

McAb	阴性对照 Negative control	阳性对照 Positive control	1∶1 000	1∶2 000	1∶4 000	1∶8 000	1∶16 000	1∶32 000	1∶64 000	1∶128 000	1∶256 000	1∶512 000
2D11H7	0.066	1.971	1.295	1.103	0.98	1.036	0.881	0.642	0.524	0.491	0.383	0.216
3G3D4	0.075	1.935	1.665	1.531	1.476	1.354	0.988	0.743	0.632	0.432	0.402	0.315
3H3C8	0.053	2.014	2.054	2.037	2.007	1.914	1.727	1.38	1.009	0.803	0.554	0.505

图4 两种引物PCR检测效果比较 1~5号泳道,1-5号EHP阳性样品用SWP2-F/R引物的扩增结果;7~11号泳道,1-5号EHP阳性样品用18S-F/R引物的扩增结果;6号泳道为DL2000分子质量标记。

Fig.4 Comparison of PCR detection with two kinds of primers Lane 1-5, PCR results to No. 1-5 positive samples with SWP2-F/R primer; Lane 7-11, PCR results to No. 1-5 positive samples with 18S-F/R primer; Lane 6, DNA marker (DL2000).

图5 Western blot检测结果 0号泳道,EHP阴性样品的杂交结果;1~5号泳道,1~5号EHP阳性样品的杂交结果。

Fig.5 Results of Western blot analysis Lane 0, Negative sample with Western blot; Lane 1-5, Results to No. 1-5 positive samples with Western blot.

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虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用

Cloning, expression and application in detection of SWP2 gene in Enterocytozoon hepatopenaei

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